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Nanogenotox: The project

Introduction

Nanotechnology is a highly strategic industrial and economic sector with enormous potential benefits for many societal and environmental domains.

"Towards a method for detecting the potential genotoxicity of nanomaterials"

Human exposure to manufactured nanomaterials (MNs) used in consumer products may occur during several phases of their life cycle, from synthesis, production and inclusion in products to the release of these nanomaterials into the environment.

The lack of scientific knowledge and the absence of evidence demonstrating the safety of certain nanotechnology products make regulation very difficult (Source: January 2009 SCENIHR opinion). Because of this, health and safety evaluation is attracting the attention of the public and of governments worldwide.

The Executive Agency for Health and Consumers awarded a grant through the second programme of Community action in the field of health (2008-2013) for a Joint Action on the "Safety of nanomaterials".

The aim of this Joint Action is to establish a robust (specific and sensitive) methodology to assess the potential genotoxicity (i.e. inducing DNA damage) of MNs and to generate data on the genotoxic effect of certain reference materials.

Project description

Title: Safety evaluation of manufactured nanomaterials by characterisation of their potential genotoxic hazard.

Priority area: Improve citizen’s health security

Context of the Joint Action:

A call for a Joint Action on "the Safety of Nanomaterials" under Section 3.2 "Improve citizens' health security" of the Community Public Health Program work program was published on Thursday, 26 February 2009, following an initial proposal by France (DGS) This Joint action was approved in July 2009 for 6.2 millions of Euros of which 2.9 M€ (46%) were funded by European Commission’s health programme, while partners and some ministries of the participating Member States (Belgium, France, Germany, and Netherlands) provide the remaining.

The Action was launched on 18-19 March at the AFSSET (Paris, France) the project aims to obtain a sound and reliable method for detecting the potential genotoxicity of manufactured nanomaterials within 3 years.

Executive summary

Nanotechnology is a highly strategic industrial and economic sector revealing enormous potential benefits for many societal and environmental domains. Human exposure to manufactured nanomaterials used in consumer products may occur during several phases of their life cycle: from synthesis, production and inclusion in the final products to the release of manufactured nanomaterials to the environment (through industrial emissions or product disposal).

Nanotoxicology, that study of the potential adverse effects of chemicals (in our case nanomaterials) on living organisms, is thus attracting the attention of the public and of governments worldwide.

The lack of scientific knowledge and absence of evidence of the health and safety potential hazards of nanotechnology products, however, make regulation very difficult (source January 2009 SCENIHR opinion pdf ) The general objective of the Joint Action is a high added-value complement to the Member States’ policies and contributes to improve citizens’ health security and also participates in the EU’s leadership position in this field as well as in ongoing international work ( OECD, ISO…).The aim of the Joint Action is highly relevant in the current context. It targets crucial items identified in the programme providing a genuine European dimension as 16 institutions from 11 Member States, including new Member States, are involved.

The methods and means implemented include 4 scientific Work Packages (WPs) with the aim of:

Strengthening, expanding and sharing the knowledge required for hazard, exposure and overall risk assessment of manufactured nanomaterials (MNs), in particular by building a strategy able to generate relevant and reliable data for EU public health authorities (scopes 1,2,3)

Accelerating the exploitation of existing data, FP6 (Sixth Frame Work Programme) and FP7 (Seventh Frame Work Programme) projects such as: NANOINTERACT, NANOSTRAND, NANOSH, NANOSAFE, and the exchange of best practices in risk assessment and management, thus minimising the potentially harmful long-term effects of MNs. The Joint Action may offer society alert signals for genotoxic substances leading to potential cancers or reproductive diseases. It constitutes a first step towards creating a future programme by public health authorities based on long-term animal studies or epidemiological population surveillance (scopes 1,2,3).

Promoting the establishment of a robust methodology to screen potentially genotoxic MNs. To achieve this, fully characterised MNs widely used in consumer products are tested with standard in vitro assays completed with specific tests. Using these results, a ring test (among participating MS laboratories) for the relevant assays is performed. This methodology may be used by regulatory control bodies and industrials to check for possible genotoxicity as alternative techniques to animal experimentation. In vivo assays are conducted to characterise the toxicokinetics of selected MNs and validate the in vitro data. Standard Operating Procedures ( SOPs)will be used and if lacking, will be established as a priority. If needed, the audit of laboratories (cross evaluation) and/or staff training is planned to ensure that the laboratories involved conduct their studies following the SOPs contributing then to highlight national reference laboratories in this field (scope 2).

Major expected outcomes of this Joint Action are:

Increasing health information and knowledge about the human and environmental safety of MNs by generating relevant and reliable data sets by:

Establishing a correlation between in vivo and in vitro geno-toxicological data and completing information on manufactured nanomaterial bioaccumulation by identifying target organs.

Promoting a robust reliable methodology for testing potential genotoxicity of MNs by exchanging best practices through a round robin test. The MNs to be tested are widely available in many kinds of products, namely TiO2 and SiO2 MNs, but also carbon nanotubes ( CNT) which, according to the literature, might present toxicological effects in experimental animals similar to asbesto

The JA will provide quick, reliable and economical tests to assess potential genotoxicity of MNs with alert signals useful for society and industries. Distinguishing specific hazards regarding the physical and chemical parameters of MNs.

General objective of the project

The general objective of the JA is to complement, support and add value to the Member States' policies and to contribute to increasing the safe use of MNs in the European Union. The JA intends to improve citizens' health security by:

(i) Strengthening, expanding and sharing the knowledge required for the assessment of the hazard, exposure and overall risk of MNs at the European level. The JA provides a genuine European dimension since it involves a significant number of institutions from many Member States. It will contribute to building a strategy able to generate relevant and reliable data for Public Health authorities to assess the risk of nanomaterials.

(ii) Accelerating the exploitation of existing data (using previous and ongoing EU FP6 and F P7 projects (e.g. NANOSTRAND, NANOSAFE, NANOSH, NANOINTERACT) and the exchange of best practices in risk assessment and management thus minimising the potentially harmful long-term effects of MNs. The JA will thus contribute to giving society alert signals for genotoxic substances. It will constitute the first step towards the creation of a future programme based on long-term animal studies or epidemiological population surveillance by Public Health authorities.

These two objectives above are implicit throughout the whole JA and its 4 scientific work packages.

(iii) Promoting the establishment of robust methodologies throughout the EU. In order to make available a robust methodology (specific and sensitive) to screen potentially genotoxic MNs, fully characterized MNs widely used in consumer products will be tested with standard in vitro assays completed with specific tests. Taking into account these results, a ring test (among the participating Member State laboratories) for the relevant assays will be performed in order to establish a robust methodology to be used by the regulatory control bodies and industries to check for possible genotoxicity using alternative techniques to animal experimentation. In vivo assays will be conducted to characterize the toxicokinetics of selected MNs and compared to in vitro data.

Target groups

Regulation on MNs is nearly absent, yet there is growing concern, as highlighted in the January 2009 SCENIHR opinion on the Risk assessment of products of nanotechnologies. Long-term risks of MNs to public health may be important, which is why priority is given to cancerogenesis of MNs for which the preliminary required step is their characterization. The partners included in the proposal were selected for their knowledge of the field, and of their skills, with due notice to the budgetary constraints.

Targets groups are:

(a) The general public. An overview of nanomaterial present in consumer products and available on the European market shows that MNs are used for a wide variety of applications (e.g. pharmaceuticals, food...) and technologies (e.g. ICT, energy, transport...). The most important product categories in Europe are: motor vehicles, electronics, computers, personal care, cosmetics and household. As for all newly developed substances or products, attention should be paid to potential health risks.

(b) The regulatory authorities and market surveillance bodies. Implicitly, according to REACH, the use of MNs is regulated by manufacturers (and importers) responsible for the safety of the chemicals or products they produce (or import), enabling the authorities to take action if products pose a health risk.

It is, however, questionable if new risks arising from the presence of MNs will be recognized through the current regulatory system. More knowledge is therefore needed to assess the extent to which the current legislations can identify potential new risks.

(c) The implicated industries which should apply the developed methodology before marketing their MNs directly or in consumer products.

(d) The policy-making bodies. Until there is an evidence base on which the nature of the risks posed by MNs can be determined, it is not possible to assess the extent to which the implementation of current legislation addresses all potential risks posed by MNs.

Methods and means

Member states who are developing their skills on this topic were also included, with special attention to their needs through the implementation of audits, and if necessary, of training activities. The framework of the JA thus includes, if needed, depending on the capacities of the laboratories involved to correctly perform the experiments according to the protocols defined, the audit of laboratories and/or staff training to ensure that the different laboratories involved run studies in their own lab with the correct SOPs.

The technical role of each partner organization is clearly defined in the work package descriptions. The partners involved are key organizations in the field within each country; this and the close links with ministries in the JA will ensure that the JA results will be communicated to participating countries. Other countries will be informed of the JA through an active dissemination strategy with a number of proposed tools, and through possibilities for further possible involvement as collaborating partners (this is already the case with the UK and Ireland). In addition, various JA partners have had the opportunity to work together in the past in research projects (co-funded by the EC or other sources) as well as in programmes such as the OECD WPMN.

To build a robust reliable methodology with alternative tests for risk assessment of MNs, the following list of prerequisites will ensure that the testing results are comparable:

Standard operating procedures (SOPs) are used and disseminated to all participants by the WP leaders including handling, dosimetry and dispersion of MNs in culture medium etc. If a SOP is lacking, this will be the first priority milestone.

As far as possible, all experimental studies will use the same origin/source of particles (i.e. same production method and physicochemical characteristics). The aim is to use the same specifically designated batch for each WP. For each study, a control of the culture media used by the different laboratories will be done to check for possible variations.

OECD guidelines will be preferably used and, if needed, adapted to take into account MN specificities.

Each WP, for the same type of work, will harmonise practices to minimise potential variations due to different batches and methods of sample preparation

The JA has 4 scientific WPs:

To interpret and compare genotoxicity tests results, the MNs need to be fully characterized (WP 4) including detailed physicochemical properties and SOPs for producing suitable suspension of the MN types in mediums used for genotoxicity testing. MNs are delivered by industry with technical specifications and dispersed following standardized procedures to be agreed. Sufficient quantities from defined and characterized batches are supplied to partners at comparable qualities for each WP.

For in vitro genotoxicity testing (WP5), OECD GD 34 needs consideration in planning of the standardized round robin tests. All routes of exposure will be covered by different cell lines: pulmonary cell lines and one intestinal for all MNs, one reconstructed human full skin model only for TiO2 due to its specific use in cosmetics. Comet and micronucleus assays will be performed as well as MLA and lymphocytes micronucleus assay following the available international guidance documents.

For in vivo genotoxicity (WP6), as far as it remains feasible, in light of technical constraints and limitations (feasibility with regards to dispersion, characterization protocols, the reproducibility of the protocols etc.), pulmonary and oral exposure routes will be tested on selected MNs, using instillation to mimic inhalation and gavage for oral exposure. Skin models will not be used because rats are not sensitive to the dermal route and the use of other animals e.g. mini-pigs raise ethical problems.

For toxicokinetics (WP7), oral route and IV routes will be considered for TiO2 and SiO2 and only IV for CNT in order to know which doses to administer to rats and identify target organs for accumulation. Selection of the MN dose-range to be tested is crucial for in vitro and animal exposure: realistic doses (epidemiologically meaningful doses) will be used.

Each scientific work package, in order to achieve the third general objective of promoting the Establishment of a robust methodology must carry out, upstream, the first 2 general objectives mentioned through studying the state-of-the-art and existing data in order to choose what is relevant for MNs and justify their choice of protocols. Furthermore, each laboratory involved in the JA must run these protocols and it is important that each step is fully defined and justified as all scientific WPs are interlinked and dependant on the actions and results of the other scientific WPs. Hence, knowledge sharing is central to the JA.

Expected outcomes

Human exposure to MNs used in consumer products may occur during several phases of the life cycle of those consumer products, i. e. the synthesis of the MNs, the production, inclusion and use in consumer products and the release of MNs to the environment (through industrial emissions or disposal of consumer products). Therefore, all exposure routes will be tested on MNs selected from industry, both in vitro and in vivo. The expected outcomes deal mainly with information that is not currently available.

The major outcomes are:

1) Collecting, analyzing and disseminating health information and knowledge about the human and environmental safety of nanomaterials (scopes 1 and 2) by generating relevant and reliable data sets by:

(ii) Providing toxicological parameters for risk assessment and better understanding the behaviour of MNs by comparing them to conventional materials (scopes 1, 2 and 3).

(iii) Establishing a correlation between in vivo and in vitro genotoxicological data as well as completing the information on MN bioaccumulation by identifying target organs (scope 2).

2) Promoting a robust reliable methodology at the European level in order to be used for testing potential genotoxicity of MNs by exchanging best practices through a round robin test involving 11 European States including New Member States (scopes 1, 2 and 3). This methodology will have t he interesting advantage of quickly defining an available economical procedure for MNs genotoxicity alert signals. In addition, these alert signals could be applied to MNs, already widely used on the market, namely TiO2 and SiO2, but also to MNs, which according to the current literature, have some toxicological effects in experimental animals, namely CNT. This action will help reinforce the European Union's leadership on MN safety and contribute to international work already ongoing ( OECD and ISO). An additional benefit may be a possible application to the non-intentionally produced nanomaterials (e.g., nanoparticles form smoke and diesel), thus providing a better understanding of their behaviour (scope 2).

Thus, the outcomes of the JA target crucial items of the programme by facilitating overall safety evaluation for MNs (scopes 2 and 3), sharing knowledge on identified MNs (scope 1) and filling the gaps in risk assessment through genotoxicity ring testing (scopes 1, 2 and 3). The outcomes also bring significant added value at the European level through the participation of key actors in the consortium.

Furthermore, national reference laboratories will be highlighted through the consistent use of standard operating procedures and cross evaluation and/or staff training in the laboratories involved.

Concerning the long term contribution or sustainability of the JA, this will be ensured in a number of ways. At a minimum, a network of reference laboratories will have been created. Those laboratories could continue, even after the JA, to work either on their own or within the network, on the basis of the developed SOPs to continue the work. In addition, a database will have been developed within the JA, with data that are valid and exploitable and can serve as a reference. These data will be available for different materials, selected for their different potential levels of toxicity (based on current knowledge), materials which are widely produced and used, available on the market, and for which exposures occur through different routes. In addition to the creation of a database, the JA will develop a robust, rapid, reliable method, alternative to animal testing, capable of giving alert signals to different target groups:

Manufacturers who may decide to stop developing materials on the basis of the obtained results, or control authorities, or citizens. The developed method can be transferred to other stakeholders outside the JA. Also, for the organizations participating in the JA, the project will offer a possibility to share technical competences as well as procedures and SOPs.